Human Milk ligosaccharides (HMO) are the third most abundant component of human milk. While accumulating evidence suggests that they benefit the breast-fed infant, little is known about how HMO are synthesized in the mother's mammy gland. Up until now, one of the critical barriers in elucidating HMO biosynthesis has been the lack of suitable models that synthesize oligosaccharides that resemble those found in human milk. We now provide preliminary results that place us in a unique position to overcome this critical barrier. We present a model based on mammary gland epithelial cells that were derived from progenitor cells isolated from human milk. Upon exposure to prolactin, these cells synthesize and secrete oligosaccharides that contain all structural features of HMO, including polylactosamine elongation and branching as well as fucosylation and sialylation in all linkages known to occur in HMO. Results are highly reproducible. Oligosaccharide composition in the cell culture supernatant and in the respective milk sample match and reflect interpersonal variations between different milk donors. Oligosaccharide composition in the cell culture media changes in response to cell manipulation with glycosyltransferase-specific small interfering RNA. To date, this is the first and currently only model available to explore HMO biosynthesis. Our application proposes to use this new powerful tool to determine which glycosylation-related genes are differentially expressed when mammary gland epithelial cells are exposed to prolactin (Specific Aim 1) and to identify which enzymes contribute to HMO elongation and branching, fucosylation and sialylation (Specific Aim 2). The proposed experiments and anticipated results have a high potential to significantly advance our knowledge and understanding on how HMO are synthesized in the human mammary gland. A better understanding of how HMO are synthesized in nature will help facilitate in vitro enzymatic and chemo-enzymatic HMO synthesis. In addition, the knowledge gained from the proposed studies may lay the groundwork to bioengineer a cell-based system that contains the glycosylation machinery required to synthesize not one, but a mix of a variety of different oligosaccharides that resemble the complex compositions of oligosaccharides naturally occurring in human milk. HMO produced in these in vitro and in vivo systems can be used for research in in vitro assays and animal models, for clinical intervention studies, and eventually to supplement formula to provide all infants with the benefits of HMO.